This invention relates to the stabilisation and storage of materials. The principal envisaged field of application is materials employed in the biochemical field and some pharmaceuticals.
A few biologically active materials (e.g. some proteins) are sufficiently stable that they can be isolated, purified and then stored in solution at room temperature. For most materials however this is not possible and some more elaborate form of stabilisation/storage procedure must be used.
A xe2x80x9crepertoirexe2x80x9d of techniques is known. Not all of them are useful for all materials that give rise to a storage problem. Known storage/stabilisation techniques which are applied to materials after isolation into an aqueous suspension or solution are:
(i) Addition of high concentration of chemical xe2x80x9cstabilizerxe2x80x9d to the aqueous solution or suspension. Typically 3M ammonium sulphate is used. However, such additives can alter the measured activity of enzymes and can give ambiguous or misleading results if the enzyme is used in a test procedure. (R. H. M. Hatley and F. Franks. Variation in apparent enzyme activity in two-enzyme assay systems: Phosphoenolpyruvate carboxylase and malate dehydrogenase. Biotechnol. Appl. Biochem. 11 367-370 (1989)). In the manufacture of diagnostic kits based on multi-enzyme assays, such additives often need to be removed before the final formulation. Such removal, by dialysis, often reduces the activity of an enzyme.
(ii) Freeze/thaw methods in which the preparation, usually mixed with an additive (referred to as a cryoprotectant) is frozen and stored, usually below xe2x88x9250xc2x0 C., sometimes in liquid nitrogen. Not all proteins will survive a freeze/thaw cycle.
(iii) Cold storage, with a cryoprotectant additive present in sufficient concentration (e.g. glycerol) to depress the freezing point to below the storage temperature and so avoid freezing. For example in the case of restriction endonucleases, the enzymes need to be protected against freezing by the addition of high concentrations of glycerol and maintained at xe2x88x9220xc2x0 C. Use of an additive in high concentration may also reduce the specificity of restriction enzymes and give rise to so-called xe2x80x9cstar-activityxe2x80x9d. (B. Polisky et al. PNAS USA, 72, 3310 (1975)).
(iv) The commonest method for the stabilisation of isolated protein preparations is freeze-drying, but this process can only be applied to freeze-stable products. The aqueous isolate of the active material in a suitable pH buffer and in the presence of a cryoprotectant is first frozen, typically to xe2x88x9240xc2x0 to xe2x88x9250xc2x0 C.; the ice is then removed by sublimation under vacuum and at low sub-zero temperatures, following which the residual moisture which may amount up to 50% of the xe2x80x9cdriedxe2x80x9d preparation is removed by desorption during which the temperature gradually rises. The complete freeze-drying cycle may take several days and is costly in capital and energy. Freeze-drying also suffers from technical disadvantages because of its irreproducibility. Suppliers of freeze-dried protein products generally specify storage at xe2x88x9220xc2x0 C. rather than ambient temperature. Exposure to ambient temperatures for periods of days to weeks can result in significant activity losses.
(v) Undercooling, as described in European Patent 0 136 030 and by Hatley et al. (Process Biochem. 22 169 (1987)) allows for the long-term (years) stabilisation of proteins without the need for additives. However, while this process extended the previous repertoire of possibilities, the undercooled preparations need to be shipped at temperatures not exceeding +5xc2x0 C. and must be stored, preferably at xe2x88x9220xc2x0 C. They also have to be recovered from a water-in-oil dispersion prior to their final use.
It will thus be apparent that a stabilisation/storage process which enabled storage at ambient temperature would be very desirable, since it would avoid the need for low temperature storage entailed by existing processes. Hitherto, however, storage at ambient temperature has been impossible for many materials.
There would also be advantage in adding to the existing xe2x80x9crepertoirexe2x80x9d of processes for stabilisation and storage, because some of the existing processes are limited in their applications or entail accepting disadvantages such as a need to mix with a stabilising agent which is difficult to remove later.
There would furthermore be advantage in providing a more cost effective process than the current freeze-drying process.
We have found, surprisingly, that materials which are not stable when isolated and held in solution at room temperature can nevertheless be successfully incorporated into a glass formed from a water-soluble or water-swellable substance, and can later be recovered. While in the glass the material is immobilised and stable.
In a first aspect this invention provides a storable composition comprising at least one material to be stored, preferably selected from the group consisting of proteins, peptides, nucleosides, nucleotides and enzyme cofactors, dissolved in a water-soluble or water-swellable substance which is in an amorphous, glassy or (much less preferably) rubbery state.
As will be explained in more detail below, it is preferred that the composition displays a glass transition temperature of at least 20xc2x0 C. preferably at least 30xc2x0 C.
It may be desirable that the compositions has a water content of not more than 4% by weight.
The invention may be utilised for stable storage of a single material, or for a mixture of materials which have little or no effect on each other.
However, in a development of this invention, a single composition contains a plurality of materials which form part or all of a reacting system. These may be fairly simple chemicals.
In a further aspect, this invention provides a method of rendering a material suitable for storage, comprising dissolving the material in a water-soluble or water-swellable substance or solution thereof and forming the resulting mixture into a glass.
This process is capable of being carried out without the use of any non-aqueous organic solvent, which is advantageous because such solvent could prove harmful to many substances. Also processing with and/or removal of organic solvents can be undesirable for environmental reasons.
A further feature is that the process is energy efficient, requiring much less energy than freeze drying. Most of the drying can be done at less than 40xc2x0 C.
The material(s) stabilized for storage may potentially be any of a wide range of materials which are ordinarily liable to undergo a chemical reaction which is dependent on diffusion of reacting species.
One category of materials to which the invention is applicable is proteins and peptides, including derivatives thereof such as glycoproteins. Such proteins and peptides may be any of: enzymes, transport proteins, e.g. haemoglobin, immunoglobulins, hormones, blood clotting factors and pharmacologically active proteins or peptides.
Another category of materials to which the invention is applicable comprises nucleosides, nucleotides, dinucleotides, oligonucleotides (say containing up to four nucleotides) and also enzyme cofactors, whether or not these are nucleotides. Enzyme substrates in general are materials to which the invention may be applied.
The material for stabilisation and storage may be isolated from a natural source, animal, plant, fungal or bacterial, or may be produced by and isolated from cells grown by fermentation in artificial culture. Such cells may or may not be genetically transformed cells.
The material will need to be soluble in aqueous solution, at least to the extent of forming a dilute solution which can be used for incorporation into the glass forming substance.
As mentioned above, a development of this invention is to store more than one component of a reacting system in a glass. This can be useful for materials which will be required to be used together in, for example, an assay or a diagnostic kit.
Storing the materials as a single glassy preparation provides them in a convenient form for eventual use. For instance, if an assay requires a combination of a substrate, or cofactor and an enzyme, two or all three could be stored in a glass in the required concentration ratio and be ready for use in the assay.
If multiple materials are stored, they may be mixed together in an aqueous solution and then incorporated together into a glass. Alternatively they may be incorporated individually into separate glasses which are then mixed together.
When multiple materials are stored as a single composition (which may be two glasses mixed together) one or more of the materials may be a protein, peptide, nucleoside, nucleotide or enzyme cofactor. It is also possible that the materials may be simpler species. For instance a standard assay procedure may require pyruvate and NADH to be present together. Both can be stored alone with acceptable stability. However, when brought together in aqueous solution they begin to react. If put together in required proportions in the glassy state they do not react and the glass can be stored.
A glass is defined as an undercooled liquid with a very high viscosity, that is to say at least 1013 Pa.s, probably 1014 Pa.s or more.
Normally a glass presents the appearance of a homogeneous, transparent, brittle solid which can be ground or milled to a powder. In a glass, diffusive processes take place at extremely low rates, such as microns per year. Chemical or biochemical changes including more than one reacting moiety are practically inhibited.
Above a temperature known in the glass transition temperature Tg, the viscosity drops rapidly and the glass turns into a rubber, then into a deformable plastic which at even higher temperatures turns into a fluid.
The glass forming substance employed in this invention must be hydrophilicxe2x80x94either water-soluble or water-swellablexe2x80x94so that water will act as a plasticiser. Many hydrophilic materials, both of a monomeric and a polymeric nature either exist as or can be converted into amorphous states which exhibit the glass/rubber transitions characteristic of amorphous macromolecules. They have well defined glass transition temperatures Tg which depend on the molecular weight and a molecular complexity of the glass forming substance. Tg is depressed by the addition of diluents. Water is the universal plasticiser for all such hydrophilic materials. Therefore, the glass/rubber transition temperature is adjustable by the addition of water or an aqueous solution.
For this invention it will generally be necessary that the glass forming substance, when anhydrous or nearly so, displays a glass transition temperature Tg in a range from 20 to 150xc2x0 C., preferably 25 to 70xc2x0 C. If Tg is towards the higher end of the range, a lower Tg can be achieved by adding water which can be removed after the material which is to be stored has been incorporated into the glass. Mixtures of glass forming substances may be used if the components are miscible as a solid solution. If so, material(s) of lower Tg serve as plasticiser(s) for material(s) of higher Tg.
If Tg of the final composition is sufficiently high, storage can be at room temperature. However, if Tg of the composition is close to or below room temperature it may be necessary or desirable to refrigerate the glassy composition if storage is for a prolonged period. This is less convenient but still is more economical than freeze-drying.
If the composition is heated above its Tg during storage, it will change to its rubbery state. Even in this condition stored materials are stable for a considerable period of time. Consequently, it may well do no harm if the temperature of the stored material is allowed to go above Tg for a limited time, such as during transportation.
If a composition is maintained above its Tg (and therefore in a rubbery condition) the storage life will be limited but still considerable and the benefit of the invention will be obtained to a reduced extent.
Conversely, if Tg of the composition is well above room temperature, the composition is better able to withstand storage at an elevated temperature, e.g. in a hot climate.
As mentioned above, Tg of the formulated composition is typically 5xc2x0 below Tg of the anhydrous glass forming substance.
The glass forming substance should be sufficiently chemically inert towards the material which is to be incorporated in it. An absolute absence of chemical reactivity may not be essential, as long as it is possible to incorporate the material, store the glass, and recover the material without serious degradation through chemical reaction.
Many organic substances and mixtures of substances will form a glassy state on cooling from a melt.
Carbohydrates are an important group of glass forming substances: thus candy is a glassy form of sugar (glucose or sucrose). The Tg for glucose, maltose and maltotriose are respectively 31, 43 and 76xc2x0 C. (L. Slade and H. Levine, Non-equilibrium behaviour of small carbohydrate-water systems, Pure Appl. Chem. 60 1841 (1988)). Water depresses Tg and for these carbohydrates the depression of Tg by small amounts of moisture is approximately 6xc2x0 C. for each percent of moisture added. We have determined the Tg value for sucrose at 55xc2x0 C.
In addition to straightforward carbohydrates, other polyhydroxy compounds can be used, such as carbohydrate derivates like sorbitol and chemically modified carbohydrates.
Another important class of glass forming substances are water-soluble or water-swellable synthetic polymers, such as polyvinyl pyrrolidone, polyacrylamide or polyethyleneimine. Here Tg is a function of the molecular weight. Both of these classes of glass forming substances are suitable for the present invention.
A group of glass forming substances which may in particular be employed are sugar copolymers described in U.S. Pat. No. 3,300,474 and sold by Pharmacia under the Registered Trade Mark xe2x80x9cFicollxe2x80x9d. This U.S. patent describes the materials as having molecular weight 5,000 to 1,000,000 and containing sucrose residues linked through ether bridges to bifunctional groups. Such groups may be alkylene of 2, 3 or more carbon atoms but not normally more than 10 carbon atoms. The bifunctional groups serve to connect sugar residues together. These polymers may for example be made by reaction of the sugar with a halohydrin or a bis-epoxy compound.
One process of rendering a material storage stable in accordance with the present invention commences from an aqueous solution of the material (which will be referred to as the active material), and a supply of the substance into which it is to be incorporated, with this substance already in an amorphous state, either glassy or rubbery.
Then a controlled amount of an aqueous solution containing the active material is incorporated into the glassy substance, thus turning it into a rubber: the materials are mixed to homogenise the glass forming substance with the active material. The rubbery form has the consistency of a dough and can be rolled or milled into a thin sheet. This rubber is then subjected to reduced pressure, possibly accompanied by moderate heat, in order to remove most of the added moisture. The final product is a glass with a glass temperature slightly, e.g. approximately 5xc2x0, below that of the pure glass forming substance. It can be kept in the form of a transparent film or ground into a fine powder or compressed into tablet form. In the glassy state (below Tg) the deterioration of the active material, by whatever mechanism, is retarded to the extent that, on practical time-scales, even substances which in their free states are extremely labile are found to possess long shelf-lives.
Full biochemical activity is maintained, but locked in, throughout this period at temperatures below Tg and can be rapidly released by resolubilization of the glass in an aqueous medium.
The glass forming substance and the amount of solution added to it are chosen so that the rubbery material
obtained from the addition is at a temperature above its Tg (or to put it another way, its Tg is below the ambient temperature) but as moisture is removed the value of Tg increases to above the ambient temperature.
Preferably the starting substance also has its Tg above ambient temperature, so that lowering of Tg on addition of aqueous solution lowers this value from above ambient to below. However, it would be conceivable to begin with a moisture-containing substance whose Tg already lies below ambient, lower it further through addition of aqueous solution of the material to be incorporated, and finally raise Tg to above ambient temperature on drying.
The amount of aqueous solution which can and should be added to form a rubbery dough may well be found by trial and error. It is likely to be not more than 5% by weight based on the glass forming substance. The steps of adding solution to form a rubbery dough and drying this back to a glassy state can be repeated to build up the concentration of active material in the glass.
If desired, the Tg value of a sample of a glass forming substance can be determined, and determined again after mixing in varying amounts of water, so as to be able to plot a graph of Tg against moisture content.
Tg values can be determined with a differential scanning calorimeter and can be detected as a point at which a plot of heat input against temperature passes through an inflection pointxe2x80x94giving a maximum of the first temperature derivative.
Vacuum applied to assist the removal of water from the rubbery composition need not be particularly hard. Suitably it is less than 90% of normal atmospheric pressure. A pressure which is 80% of normal atmospheric pressure has been found adequate. A harder vacuum may be employed, however, if this is found convenient.
Heating of the doughy mixture to remove moisture may be at a temperature not above 80xc2x0, and for a protein is preferably not above 60xc2x0 C. Heating may not be necessary: evaporation of moisture under reduced pressure may proceed to a sufficiently low moisture content even at room temperature of around 20xc2x0 C., but of course heat accelerates the evaporation.
Another process for rendering material storage stable in accordance with the present invention can enable the material to be stored and recovered at a greater concentration of active material relative to the carrier substance. In this process a quantity of the carrier substance, or a solution thereof, is added to a solution of the active material. When the added carrier substance has dissolved fully, the solution may be divided into convenient portions, e.g. 0.1 to 1 ml. The samples of solution are placed under reduced pressure so that water is evaporated from them until the carrier substance is in a glassy state. Typical conditions are to commence the evaporation at a temperature not exceeding 40xc2x0 C., preferably in the range from 20 to 30xc2x0 C. and continue it for some hours, for instance 24 to 36 hours. As evaporation continues the glass temperature of the residual material rises. Evaporation for the period indicated can be sufficient to achieve a glass transition temperature exceeding 30xc2x0 C. Once such a sufficiently high glass transition temperature has been achieved the temperature may be raised while evaporation continues. For instance once the glass transition temperature has reached a level of 30xc2x0 C. the temperature may be raised to within a range of 40 to 70xc2x0 C., e.g. 60xc2x0 C. for a shorter time such as two hours. For this procedure also, vacuum used to bring about evaporation of water does not need to be particularly hard. It may also be found that heating is unnecessary: evaporation without heating for an extended time may achieve a sufficiently low moisture content.
In the above, the carrier substance may be added in a dry state, e.g. a powder, or as a solution.
Recovery (i.e. reactivation) of stored material can be effected by simply adding water or aqueous solution to a quantity of the glass with the active material therein. If the carrier substance is water-soluble the result is a solution of the material and the carrier substance.
Separation by chromatography to isolate the stored, active material from the glass forming substance is possible. However, in general it will be neither desirable nor necessary. Instead the glass forming substance is chosen so that it will not interfere with the use (e.g. assay) of the stored, active material.
In the case of a water-swellable glass forming substance, it will remain out of solution, perhaps as a gel, and the solution of the material can be separated by centrifugation if required.
The suitability of an intended glass forming substance and conditions for incorporation of material into it can both be checked by preparing a glass with the material incorporated, and then recovering the material without any substantial period of storage.
Storage stability can, if desired, be tested by storage at a higher temperature such as 35xc2x0 C. or even 50xc2x0 C. which gives an accelerated test.